1. Field of Invention
The present invention relates generally to health care diagnostics and specifically to an improved method for oral sampling in a human subject for rapid detection of the presence of infection, the specific embodiment being the detection of the urease activity associated with Helicobacter pylori infection in the human.
Helicobacter pylori (H. pylori) is a common pathogen in humans and specifically causes disease of the stomach. In industrialized countries, infection can be present in half of all persons older than 50 years. First identified in 1983, H. pylori is now known to cause chronic gastritis or inflammation of the stomach, as well as gastric and duodenal ulcers. It is also associated with gastric malignancy (Thomas, E., et al., xe2x80x9cThe role of the oral cavity in Helicobacter pylori infection,xe2x80x9d Am. J. Gastroenterol., 1997 December; 92(12): 2148-2154). This and other publications cited are incorporated by reference herein.
Combinations of antibiotics and bismuth and/or gastric acid blocking agents are used to treat H. pylori infection in the stomach. Bismuth salicylate inhibits the urease activity of H. pylori (Prewett, E., et al., xe2x80x9cComparison of one-day oral dosing with three bismuth compounds for the suppression of Helicobacter pylori assessed by the 13C-urea breath test,xe2x80x9d Aliment. Pharmacol. Ther., 1992 February; 6(1): 97-102).
The scientific literature states that the mode of transmission of H. pylori between humans is unknown. H. pylori has been identified in the mouths of humans in some instances, involving isolation from the dental plaque, saliva and gingival pockets. However, studies of the prevalence of oral colonization by H. pylori have shown variable results with most known infected persons having negative oral results (Thomas et al., 1997, supra).
In some instances an association has been observed between H. pylori detectable in the mouth and presence of the organism in the stomach (Mapstone, N., et al., xe2x80x9cIdentification of Helicobacter pylori DNA in the mouths and stomachs of patients with gastritis using PCR,xe2x80x9d J. Clin. Pathol., 1993; 46: 540-543). In some cases the strain of H. pylori in saliva is found to match that in the stomach (Ferguson, D., et al., xe2x80x9cIsolation of Helicobacter pylori from saliva,xe2x80x9d J. Clin Microbiol., 1993 October; 31(10): 2802-2804). In lieu of oral colonization, it has been suggested that reflux of gastric contents (gastroesophageal reflux) into the oral area may be the factor accounting for occasional recovery of H. pylori organisms from the mouth (Madinier, I., et al., xe2x80x9cOral carriage of Helicobacter pylori: a review,xe2x80x9d J. Periodontol., 1997 January; 68(1): 2-6).
H. pylori has been suggested to be associated with health disorders beyond gastric inflammatory conditions (Wisniewski, R., and Peura, D., xe2x80x9cHelicobacter pylori: behond peptic ulcer disease,xe2x80x9d Gastroenterologist, 1997 December; 5(4): 295-305; xe2x80x9cThe germ theory of heart disease,xe2x80x9d Newsday, Mar. 3, 1998; Markus, H., and Mendall, M., xe2x80x9cHelicobacter pylori infection: a risk factor for ischemic cerebrovascular disease and carotid atheroma,xe2x80x9d J. Neurol. Neurosurg. Psychiatry, 1998 January; 64(1): 104-107).
Detection of H. pylori in oral fluid samples derived from the pharynx, as an indicator of current infection status has not been described in humans until the present invention. In one study, tissue biopsies of the larynx, which lies below the pharynx and is not contacted during sampling of the pharynx, were found to contain urease activity in only a minority of subjects with chronic laryngitis (Borkowski, G., et al., xe2x80x9cA possible role of Helicobacter pylori infection in the etiology of chronic laryngitis,xe2x80x9d Eur Arch Otorhinolaryngol, 1997; 254: 481-482).
Some research suggests that conditions of the pharynx or throat area may be involved in diseases of the gastrointestinal tract (Zavadiak, H., xe2x80x9cThe relationship of duodenal peptic ulcer and gastroduodenitis to a chronic staphlococcal infection,xe2x80x9d Lik Sprava, 1993 May; 5-6: 65-69; Minocha, A., et al., xe2x80x9cIs a history of tonsillectomy associated with a decreased risk of Helicobacter pylori infection?xe2x80x9d, J. Clin. Gastroenterol., 1997 December; 25(4): 580-582). These studies suggest associations between changes in the lymphatic tissues and subsequent gastrointestinal disease, but not pharyngeal sampling for diagnostic purposes.
In a canine study, multiple tissue biopsies were obtained following experimental H. pylori infection. H. pylori was found in a pharynx biopsy of only one of several experimentally infected, gnotobiotic canine test subjects (Radin, M., et al., xe2x80x9cHelicobacter pylori gastric infection in gnotobiotic beagle dogs,xe2x80x9d Infect. Immun., 1990 August; 58(8): 2606-2612). Canines are not naturally affected by H. pylori infection and these and other non-humans do not serve as a reservoir of H. pylori, however.
H. pylori produces abundant urease, more than other currently known bacteria. The high urease activity of H. pylori allows it to survive in an acid environment, by production of ammonia from urea and thereby signicantly elevating the pH of the environment of the organisms. The unique ability of H. pylori to produce abundant urease has been utilized to identify presence of the organism in solid tissue specimens placed into small test volumes containing urease substrate. In an initially acid pH environment of greater than approximately 2.5, the urease activity of H. pylori in a solid tissue specimen can metabolize urea to raise the pH (U.S. Pat. No. 4,923,801, Marshall and Guerrant, 1990). This and other patents cited are incorporated by reference herein. When present in significant quantities, H. pylori organisms have sufficient preformed urease to raise the pH in the absence of additional bacterial growth.
The urease activity of common, non-H. pylori bacteria is destroyed by acid environments. A relatively stronger acid environment is required to destroy the urease of H. pylori. Increased acidity stimulates the urease activity of H. pylori (Miederer, S., Grubel, P., xe2x80x9cProfound increase of Helicobacter pylori urease activity in gastric antral mucosa at low pH,xe2x80x9d Dig. Dis. Sci., 1996 May; 41(5): 944-949).
The rapid urease test has a high sensitivity for detection of urease activity and is considered a reliable method for assessment of H. pylori infection in solid tissue specimens. One rapid form of the urease test utilizes a gel into which the tissue sample is placed (CLOtest; U.S. Pat. No. 4,748,113, Marshall, May 1998). The gel acts as a support for the tissue specimen and reagents and contains the urease substrate and pH indicator. This test requires up to 24 hours for completion. Methods of applying urea and a pH indicator to gastric mucosa during endoscopic procedures, to rapidly detect tissue colonized by H. pylori are also in use (Thillainayagam, A., et al., xe2x80x9cDiagnostic efficiency of an ultrarapid endoscopy room test for Helicobacter pylori,xe2x80x9d Gut, 1991 May; 32(5): 467-469; Iseki, K., et al., xe2x80x9cHelicobacter pylori infection in patients with early gastric cancer by the endoscopic phenol red test,xe2x80x9d Gut, 1998; 42: 20-23).
A test-strip form of the rapid urease test which provides visual results on solid tissue specimens within an hour or more at room temperature has been developed (Yousfi, M., et al., xe2x80x9cComparison of Agar Gel (CLOtest) or Reagent strip (PyloriTek) rapid urease tests for detection of Helicobacter pylori infection,xe2x80x9d Am. J. Gastroenterol., 1997 June; 92(6): 997-999; Elitsur, Y., et al., xe2x80x9cProspective comparison of rapid urease tests (PyloriTek, CLOtest) for the diagnosis of Helicobacter pylori infection in symptomatic children: a pediatric multicenter study,xe2x80x9d Am. J. Gastroenterol., 1998 February; 93(2): 217-219; U.S. Pat. No. 5,314,804, Boguslaski and Carrico, May 1994; U.S. Pat. No. 5,420,016, Boguslaski and Carrico, May 1995). This test strip was developed for detection of urease activity in solid tissue specimens obtained from the stomach.
2. Discussion of Prior Art
Confirmation of H. pylori infection is necessary to ensure appropriate therapy. Serological test methods are available which detect antibodies to H. pylori. These tests fail to identify a proportion of persons with gastric disease (Vaira, D., et al., xe2x80x9cUsefulness of serology in preendoscopic screening. The Italian Helicobacter pylori Study Group,xe2x80x9d Helicobacter, 1997 July; 2 Suppl. 1: S38-S43). Also, detection of salivary antibodies to H. pylori has been shown to have only limited utility for diagnosis of H. pylori infection (Loeb, M., et al., xe2x80x9cEvaluation of salivary antibodies to detect infection with Helicobacter pylori,xe2x80x9d Can. J. Gastroenterol., 1997 July; 11(5): 437-440).
Ideally, body sampling methods used for identification of H. pylori organisms in human tissues would have ease of performance, efficiency, cost-effectiveness and adequate safety. Prior art methods of detecting H. pylori infection have important limitations. As discussed, one method of identification of H. pylori infection requires invasive tissue sampling by endoscopic procedures. Once obtained, the tissue samples are subjected to methods to detect H. pylori, such as involving detection of urease activity in tissue samples. Another method of diagnosis requires stool sampling coupled with laboratory analysis to detect H. pylori antigen in fecal matter. The drawbacks of these methods are they require specially-trained personnel and specialized equipment to perform. Also, potential health risks may be associated with invasive procedures where such are required for diagnosis.
Sampling of saliva or plaque in the oral cavity has been tested as a non-invasive means of detecting H. pylori infection, however it has been rejected as being unsatisfactory for diagnosis. In the prior art, there has been only a low probability of identifying H. pylori organisms from oral samples of persons with known gastric infection (Thomas et al., 1997, supra). Prior art methods of oral sampling for H. pylori have required sophisticated laboratory techniques such as identifying genetic material of the bacteria (e.g., polymerase chain reaction). See also Husson, M., et al., xe2x80x9cDetection of H. pylori in saliva using a monoclonal antibody,xe2x80x9d Int. J. Med. Microbiol. Virol., Parasitol. Infect. Dis., 1993 November; 279(4): 466-471.
The medical literature continues to express the opinion that the mode of transmission of H. pylori infection in humans is unknown and that H. pylori is only occasionally cultured from saliva in persons with known infection (Parsonnet, J, et al., xe2x80x9cFecal and oral shedding of Helicobacter pylori from healthy infected adults,xe2x80x9d JAMA, Dec. 15, 1999; 282(23): 2240-2245). The above study used induced vomiting and diarrhea to obtain gastrointestinal samples for testing. Thus the prior art teaches away from use of oral fluid sampling for diagnosis of H. pylori infection.
Another factor is that normally present oral bacteria can inhibit growth of H. pylori (Ishihara, K., et al., xe2x80x9cOral bacteria inhibit Helicobacter pylori growth,xe2x80x9d FEMS Microbiol. Lett., Jul. 15, 1997; 152(2): 355-361). Thus it would be surprising that oral sampling could be used for diagnosis of H. pylori infection.
A method has been described for xe2x80x9chomexe2x80x9d detection of infection, however it requires incubation of saliva samples in selective growth media, over a period of days to enable possible detection of H. pylori present in such samples (U.S. Pat. No. 5,498,528, King, March 1996). Detection of urease activity as an indicator of H. pylori is used in gastric biopsies as a rapid test for infection, however this is based on high levels of organisms present in gastric infection. A similar approach to rapid detection of H. pylori using oral samples would fail, based upon prior art.
Still a further problem with oral sampling is that organisms which are native to the oral cavity have urease activity (Dibdin, G., Dawes, C., xe2x80x9cA mathematical model of the influence of salivary urea on the pH of fasted dental plaque and on the changes occurring during a cariogenic challenge,xe2x80x9d Caries Res., 1998; 32(1): 70-74). Contamination by these urease-producing organisms in the mouth can cause false-positive results when attempting to use the urease method to test tissue samples for H. pylori infection (Namavar, F., et al., xe2x80x9cPresence of Helicobacter pylori in the oral cavity, oesophagus, stomach and faeces of patients with gastritis,xe2x80x9d Eur. J. Clin. Microbiol. Infect. Dis., 1995 March; 14(3): 234-237; Marshall, B., and Surveyor, I., xe2x80x9cCarbon-14 urea breath test for the diagnosis of Campylobacter Pylori Associated Gastritis,xe2x80x9d J. Nucl. Med., 1988 January; 29(1): 11-16; Surveyor, I., et al., xe2x80x9cThe 14C-urea breath-test for the detection of gastric Campylobacter pylori infection,xe2x80x9d Med. J. Aust., 1989 October; 151(8): 435-439; Marshall, B., et al., xe2x80x9cA 20-minute breath test for Helicobacter pylori,xe2x80x9d Am. J. Gastroenterol., 1991 April; 86(4): 438-445).
In addition, urea is normally present in saliva as a result of its production in the body (Dibdin and Dawes, 1998, supra) and is used as a substrate by the urease-producing bacteria normally present in the mouth. Therefore, even without an external urea source, oral samples can demonstrate urease activity and ammonia production. Acid pH (below 5.0) has been found to inhibit urease activity of oral bacteria including Streptococcus salivarius (Sissons, C., and Hancock, E., xe2x80x9cUrease activity in Streptococcus salivarius at low pH,xe2x80x9d Arch. Oral Biol., 1993 June; 38(6): 507-516), whereas the urease activity of H. pylori is active well below this pH.
Application of acidification of oral samples for selection and rapid detection of the urease activity of H. pylori has not been previously described. Furthermore, in light of the prior art which concludes that oral sampling is not effective for H. pylori diagnosis, or requires incubation of oral samples, it would not be anticipated that acidification of oral samples would have any utility in rapid H. pylori diagnosis. Thus it would be surprising to diagnose H. pylori infection in humans by selection and rapid detection of urease activity in oral samples. The latter method depends upon oral presence of viable H. pylori and its associated urease activity, to a degree sufficient to enable rapid and selective detection without culturing or sophisticated laboratory methods.
It is the surprising discovery of this invention that it is possible to rapidly detect the urease activity associated with H. pylori directly in oral liquid samples obtained in a non-invasive, non-instrumented manner and without need for sample incubation. Further, it is possible to selectively detect the urease activity associated with H. pylori in such samples by sample acidification.
Urease activity associated with H. pylori refers to urease activity in an oral liquid sample that remains intact with acidification to a pH in a range of about 5.0 to about 2.5. Selective detection of urease activity refers to detection of the urease activity associated with H. pylori infection, with other bacterial sources of urease not being detected.
Oral liquid samples refer to samples that are collected by the process of using water or other suitable sampling liquid to contact oral tissues, followed by retrieval of the resulting oral liquid sample for direct and rapid detection of urease activity within the sample. Rapid detection refers to test results that are provided within 1 to 2 hours. Direct detection refers to testing within the container used to retrieve and hold the sample.
Non-invasive in this instance refers to performance of diagnosis without penetration of the skin or organs. Non-instrumented in this instance refers to methods of test specimen retrieval and testing without placing one or more instruments in contact with tissue surfaces.
It is a surprising discovery of this invention that the urease activity associated with H. pylori infection can be detected in oral liquid samples derived from the pharynx by the process of gargling. Retrieval of oral liquid samples from the pharynx for attempted detection of urease activity associated with H. pylori has not been previously described. It is yet a further surprising discovery and embodiment of this invention that identification of infection by detection of urease activity in oral samples is aided by sampling of both the pharynx as well as the oral cavity or mouth.
The mouth refers to the anatomic area bounded by the cheeks, lips and arch of the palate and inclusive of the teeth and tongue. The pharynx refers to the anatomic area behind and below the mouth, commonly referred to as the throat area and separated from the mouth by the space represented by the fauces.
The process of gargling involves taking sampling liquid in the lower pharynx or throat and forcing expired breath through it while holding the head back, without intentional swallowing. The process of rinsing involves taking sampling liquid in the mouth and swishing the liquid around the tissues of the mouth, including against the gingiva and over the tongue.
Sampling of both the pharynx and mouth is discovered to be advantageous for detection of urease activity associated with H. pylori. Using the methods of the invention it was discovered that such activity may be detectable either in the mouth or pharynx in a human by sampling, but not necessarily in both anatomic locations at any given point in time. These anatomic locations are distinctly sampled by the processes of rinsing the mouth and gargling, respectively. Rinsing the mouth samples saliva and plaque as well as tissues in the oral cavity such as the tongue, while gargling samples tissues in the pharynx.
Furthermore, it is discovered that in a human, the location of such urease activity may change between mouth and pharynx over time, therefore effective oral sampling for infection at a point in time includes this combination of tissues. This phenomenon has not previously been described and as noted, prior art attempts at oral liquid sampling for H. pylori detection have been limited to the mouth. The significant limitations of the prior art of oral sampling for H. Pylori are thus overcome by the present invention.
Using the methods of the invention, the unanticipated observation was made that application of a urease detecting pad containing urease substrate onto a retrieved oral liquid sample of relatively large volume, e.g., 5 to 10 ml or more, also results in measurable alteration (increase) of sample pH. This pH alteration specifically occurs when the sample contains urease activity associated with H. pylori as demonstrated by a positive test result in a urease detecting pad in contact with the sample. A urease detecting pad refers to a pad containing urease substrate that is placed in contact with the oral liquid sample.
This observation is surprising, since in the prior art of urease detection a gastric or other solid tissue specimen directly colonized with H. pylori is placed into a urease indicator. Urease activity is detected by change in pH of a small area or volume, e.g., a fraction of 1 ml surrounding the tissue specimen. Further, such specimens may be incubated and retained for a prolonged period before final test result determination.
In contrast, in the present invention any H. pylori organisms potentially retrieved by oral sampling, without the benefit of actual colonized tissue specimens are dispersed in a considerably larger volume further diluted by the sampling liquid. Yet surprisingly, this volume of oral liquid sample is capable of demonstrating significant pH change (e.g., from below 5.0 to greater than 7.0), within 1 to 2 hours and without sample incubation, when urease activity is present in the sample. Further surprisingly, this change in oral liquid sample pH is not observed when urease substrate is mixed into such oral samples, in lieu of the invention""s method of application of the urease detecting pad onto the sample for test purposes.
It is therefore an object of this invention to provide a method for rapid, specific detection of the urease activity associated with H. pylori infection in humans, directly in human oral liquid samples without the need for incubation.
It is a further object of this invention to provide a method for oral sampling and specific, rapid detection of the urease activity associated with H. pylori infection in humans, which can potentially be performed by the layperson on him/herself without professional assistance.
The foregoing and other objects, advantages and characterizing features will become apparent from the following description of certain illustrative embodiments of the invention.